Human tumorigenesis is a complicated process marked by a loss of the cell's ability to regulate gene expression and hence cell growth. As our understanding of biological mechanisms pertaining to human tumorigenesis becomes more sophisticated, it has become evident that structural studies are necessary to understand regulated processes such as DNA replication, transcription, RNA processing, and translation, which all require the coordinated action of very large multi-subunit enzymes and protein complexes. Structural biologists have been reluctant to tackle human mega-Dalton sized protein complexes due to the inability of obtaining amounts of homogeneous sample sufficient for conventional structural studies. This research proposal will implement a variety of methods to probe the structure/function relationships of large multi- subunit assemblies and address the problems associated with structural analysis of heterogenous complexes. The transcription machinery provides an ideal model system for developing the tools for structural studies of large complexes involved in human tumorigenesis. In eukaryotes, at least 80 proteins have been implicated in establishing a transcription pre-initiation complex sufficient to recognize a promoter and initiate high levels of regulated transcription. This transcription pre- initiation complex has a molecular weight in excess of 4MDa and is composed of a number of smaller stable multi-subunit complexes such as PBAF (13 subunits, 2 MDa), CRSP/ARC(16-19 subunits, 1.25-2 MDa), TFIID (15 subunits, 1MDa), RNA polymerase II (14 subunits, 0.5 MDa) and TFIIH (9 subunits, 0.5 MDa). A major objective of this P01 is to develop methods for generating more functionally and structurally homogenous samples of large multi-subunit transcription complexes with the eventual long term goal of attempting to crystallize some of these mega-Dalton sized complexes.